| ObjectiveTo explore the expression of CD gene in SK-HEP-1 cell after thermotherapy with tissue specific 5-FC,and the inhibition effect of thermot- herapy with CD/5-FC system on liver carcinoma.MethodsPlasmid PCD2 and G1AFPCDNa were amplified in competence,PCD2 and G1AFPCDNa were extracted from bacteria by the means of plasmid alkaline; PCD2 was concised by enzyme BamH 1 and EcoR 1; G1AFPCDNa was concised by enzyme Sal 1,its sequence was determined at the time.Then Plasmid PCD2 and G1AFPCDNa were transfeced into human colon carcinoma SK-HEP-1 cell respectively through liposome technique, The cells were selectively cultured with 1mg/ml G418 until positive clones were obtained; Expression of G1AFPCDNa and pCD2 mRNA in human colon carcinoma SK-HEP-1 cell were analysed by RT-PCR respectively. SK-HEP-1-G1AFPCD and SK-HEP-1- PCD2 were treated by thermotherapy with 5-FC in cell culture tube, living cell ratio was evaluated by MTT,and cell death ratio was mensured; finally,we analyze their biological activity with 5-FC in vitro respectively;we collect the rema- nent SK-HEP-1-G1AFPCD after thermotherapy,total RNA of the remanent SK-HEP-1-G1AFPCD was extracted, the expression of G1AFPCDNa mRNA in colon cancer cell SK-HEP-1 was analysed by Real -Time RT-PCR before and after thermotherapy. thirty nude mice were divided into 3 groups randomly. which were the control group,AFPCD thermotherapy group and AFPCD non-thermotherapy group; and there were 10 nude mice in each group.The mice in the control group were injected AFPCD-SK-HEP-1 cells in the spleen and NS into the peritoneal cavity.The mice in other groups were all injected AFPCD-SK-HEP-1 cells in the spleen.43℃5-Fc 500mg/kg were injected into the peritoneal cavity of the mice in the AFPCD thermotherapy group,and 5-Fc 500mg/kg were injected into the peritoneal cavity of the mice in the AFPCD non-thermotherapy group with no heating.Liver metastasis rate.the number of Iiver metastasis,the pathological changes of the tumor tissues under the electronic microscope,tumor cell apoptotic lndex andIife span and other indexes of nude mice in four groups were analyzed.The expression of CD gene in tumor tissues was detected by RT-PCR,and we use RT-PCR to confirm whether there was CD gene expression in other tissues or not,such as pancreatic gland , stomach and colon.ResultsPlasmid PCD2 and G1AFPCDNa were concised into 1.5kb and 1.9kb fragment by enzyme respectively,the sequence of G1AFPCDNa was in accordance with GenBank;the expression of plasmid PCD2 and G1AFPCDNa were certificated by RT-PCR; the expression of G1AFPCDNa were more stronger than that of PCD2; thermotherapy with 5-FC exhibited SK-HEP-1 cell transfected with G1AFPCDNa gene were more sensitive than pCD2,and the IC50 of 5-FC were 0.95mmol/l and 0.15mmol/l for each of the kind of the cell. The expression of CD gene was detected by Real-Time RT-PCR,and the ct value of CD gene expression before thermochemotherapy was 29.53±1.1974, beta-actinct was 20.82±2.7385; the ct value of CD gene expression after thermochemotherapy was 30.285±1.201, beta-actinct was 21.225±2.237;There was no difference between the two grope calculated by△△Ct relative quantitation. AFPCD-SK-HEP-1 cells were injected i in the spleen of mice of each group,and NS, 5-Fc, 43℃5-Fc were injected into the peritoneal cavity of each group at the same time,after 4 weeks,there were 10,8 and 3mice formed cancer for control group,AFPCD thermotherapy group and AFPCD non-thermotherapy group respectively;the metastasis of each grope was 100%,80%and 30%,there was no difference between control group and non- thermotherapy group(P>0.05),but there was conspicuous difference betw- een AFPCD thermotherapy group and control group(P<0.01=;the number of tumors in the liver of mice were 13.6±8.79, 5.6±2.85 and 0.6±0.65 for each of the grope, there was also conspicuous difference between AFPCD thermotherapy group and control group(P<0.01=;in common pathology, tumor cells were active, tumor giant cell and karyolobic cells were universal in control group,but in non-thermotherapy group and AFPCD thermotherapy group, tumor cells were rare, nucleus crimpled and disappeared, specifically for the AFPCD thermoth- erapy group,there were no tumor cells, fibroblast cells, lymphocyte and eosinophile granulocyte can be seen everywhere;while in the transmission electron microscope(TEM), tumor cells were difformed, Karyomegaly and evident in nucleolus, there was no apoptotic body;but in non-thermotherapy group and AFPCD thermotherapy group, tumor cells were condensed, karyopyknosis and karyorrhexis;especially for the AFPCD thermotherapy group, there were apoptotic body everywhere.The expression of CD gene can be found in each group of the tumor tissue,but there was no expression of CD in pancreatic gland , stomach and colon.ConclusionThe main conclusions of the in vitro and in vivo experimental studies areas follows:1. Plasmid PCD2 and G1AFPCDNa were transfeced into human liver carcinoma SK-HEP-1 cell respectively through liposome technique successfully, and can express themselves in SK-HEP-1 cell.2. The activity of CD gene is stronger in SK-HEP-1-G1AFPCD than in SK-HEP-1- PCD2,it can be seen that AFP TRS can drive CD gene to express greatly.3. Thermotherapy with 5-FC exhibited SK-HEP-1 cell transfected with G1AFPCDNa gene were more sensitive than pCD2, it can also be seen that AFP TRS can drive CD gene to express greatly.4. No Significant variety of CD gene expression can be detected after thermochemotherapy with 5-FC, thermochemotherapy with 5-FC can not affect the expression of CD gene in SK-HEP-1-G1AFPCD cell.5. The oncogenicity of SK-HEP-1-G1AFPCD cell is the same with SK-HEP-1 cell.6. The combined effect in the AFPCD thermotherapy group worked more markedly,and there was mutual effect between thermotherapy and CD/5-FC. Synergistic effect was acquired combining thermotherapy with CD/5-Fc system and it inhibited the formation of liver carcinoma eficiently. 7. Thermochemotherapy combined with CD/5-Fc system has specific effect on liver carcinoma,but has little influence on other tissues,such as pancreatic gland , stomach and colon. |
PDF Full Text Request